Use of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] for the treatment of cancer

ABSTRACT

IT-139, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], is an intravenously administered small molecule compound. In preclinical anti-tumor and mechanism of action studies, IT-139 showed activity against a broad range of tumor types, including those which are resistant to standard anti-cancer agents (e.g., platinums, vinca alkaloids, taxanes, anthracyclines). This activity is believed to arise from IT-139&#39;s novel mechanism of action that targets the GRP78 pathway. It was found that up-regulation of GRP78 is a key cancer cell survival pathway. Downregulation of GRP78 using IT-139 removes this resistance pathway allowing for chemotherapy and immuno-oncology agents to be more effective in treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a § 371 national stage of PCT InternationalApplication No. PCT/US2017/020209, filed Mar. 1, 2017, claiming thebenefit of U.S. Provisional Application No. 62/301,786, filed Mar. 1,2016, the contents of each of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

This invention generally relatestrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and its use in thetreatment of cancer.

BACKGROUND OF THE INVENTION

Many advances have been made in the treatment of cancers in recentyears. However, in most instances of metastatic disease, treatment isnot curative because tumor cells develop mechanisms to overcome andsurvive the damage caused by the anti-cancer agent. Targeting andovercoming these survival/resistance mechanisms of the tumor cell is anarea of anti-cancer targeting that is the subject of active research.Accordingly, there remains an unmet need to develop therapeutics totreat cancer, and, in particular, resistance.

SUMMARY OF THE INVENTION

It has now been found that compounds of the present invention, andcompositions thereof, are useful for treating cancer, and particularly,are useful for targeting survival and resistance mechanisms of tumorcells.

More specifically, it has now been found that IT-139 suppresses thestress up-regulation of GRP78 in tumor cells. This effect is specific totumor cells, as IT-139 does not affect GRP78 expression in normal cells.Treatment of normal cells under non-stressed and stressed conditionswith IT-139, showed that: 1) IT-139 does not effect the basal GRP78levels in non-stressed normal cells; and 2) IT-139 does not effect GRP78up-regulation due to stress in these same normal cells. Therefore it isbelieved that IT-139 does not impact GRP78 levels in normal cellsregardless of stress conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts GRP78 protein levels before and after treatment withIT-139 in: A) unstressed cells; and B) cells stressed with thapsigargin.

FIG. 2 depicts GRP78 mRNA levels before and after treatment with IT-139in: A) unstressed cells; and B) cells stressed with thapsigargin.

FIG. 3 depicts results from a subcutaneous syngeneic model followingtreatment with IT-139 and a PD-1 antibody.

FIG. 4 depicts transmission electron microscopy images of HCT116 cellsin: A) controlled, untreated cells; and B) HCT116 cells treated withIT-139.

FIG. 5 depicts effects of IT-139 on RNA Polymerase II Binding to GRP78promoter in: A) chip seq results; B) quantification of Pol II primers bygel electrophoresis; and C) quantification of GRP78 primers.

FIG. 6 depicts immunohistochemistry staining of HT-29 tumors (ex vivo)treated with: A) saline; and B) IT-139.

FIG. 7 depicts treatment of kidney 293T cells in stressed andnon-stressed conditions, with and without IT-139 treatment.

FIG. 8 depicts treatment of kidney 293T cells in stressed andnon-stressed conditions, with and without IT-139 treatment with respectto: A) GRP78 mRNA levels; and B) relative GRP78 mRNA expression.

FIG. 9 depicts effects of mitochondria potential following treatmentwith IT-139 in multiple cell lines.

FIG. 10 depicts cell viability of normal peripheral blood mononuclearcells following treatment with IT-139.

FIG. 11 depicts cell viability of normal peripheral blood mononuclearcells following treatment with IL-2 or IL-2 and IT-139.

FIG. 12 depicts expression of GRP78 protein levels treated with DMSO;150 IT-139; 1 μM Thapsigargin (Tg); simultaneous treatment of 150 μMIT-139 and 1 μM Tg; 1 Tg treated for 6 hours followed by 150 μM IT-139for 24 hours; and 150 mM IT-139 for 24 hours followed by 1 mM Tgtreatment for 24 hours for lanes 1-5, respectively. Lanes 6-12 are thesame treatments incubated for a further 24 hours.

FIG. 13 depicts ASPC20 cells treated in vitro for 48 hours with DMSO(control); 150 μM IT-139; 5 μM gemcitabine; simultaneous 150 μM IT-139and 5 μM gemcitabine; 5 gemcitabine for 24 hours followed by 150 μMIT-139; and 150 μM IT-139 followed by 24 hrs 5 μM gemcitabine.

FIG. 14 depicts PANC-1 cells treated in vitro for 48 hours with DMSO(control); 150 μM IT-139; 5 μM gemcitabine; simultaneous 150 μM IT-139and 5 μM gemcitabine; 5 gemcitabine for 24 hours followed by 150 μMIT-139; and 150 μM IT-139 followed by 24 hours 5 μM gemcitabine.

FIG. 15 depicts median tumor volume of 8 treatment groups in an A20Mouse model.

FIG. 16 depicts a scatter plot showing median % of Effector T Cells intumor.

FIG. 17 depicts a scatter plot showing median % of Regulator T Cells intumor.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. GeneralDescription

IT-139, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], is anintravenously administered small molecule compound. IT-139 is also knownas KP1339 or NKP1339. In preclinical anti-tumor and mechanism of actionstudies, IT-139 showed activity against a broad range of tumor types,including those which are resistant to standard anti-cancer agents(e.g., platinums, vinca alkaloids, taxanes, anthracyclines). Thisactivity is believed to arise from IT-139's novel mechanism of action,targeting the GRP78 pathway.

GRP78 (glucose regulated protein 78), also referred to as BiP or HSPA5.is a master-regulator of the endoplasmic reticulum (ER) stress response.It is also plays a critical role in tumor cell survival, anti-apoptosisand therapeutic resistance. In normal cells, GRP78 is found at lowlevels and located in the lumen of the endoplasmic reticulum. Instressed cells, GRP78 is significantly up-regulated and also foundoutside the ER in the cell cytoplasm, the nucleus, in the mitochondria,on the cell surface and secreted. The elevation of GRP78 expression in awide variety of cancer types has been correlated with increased tumorcell proliferation, metastasis, angiogenesis, and tumor cell survivaland resistance. High levels of GRP78 protein have been correlated withresistance to agents such as cisplatin, 5-FU, temozolomide, vinblastine,paclitaxel, bortezomib, sorafenib, camptothecin, etoposide, anddoxorubicin. Furthermore, treatment of tumor cell lines with several ofthese agents results in additional up-regulation of GRP78 protein. Incontrast to these anti-cancer drugs, IT-139 suppresses GRP78up-regulation in tumor cells. IT-139 suppresses GRP78 transcription.This suppression is selective to tumor cells and is most pronounced intumor cells under stress. IT-139 has no effect on GRP78 levels in normalcells whether under non-stressed or stressed conditions. As GRP78up-regulation is one of the key causes of resistance, IT-139 wasexpected to show synergy when combined with other anti-cancer agents.Preclinical studies show that IT-139 has marked synergy when used incombination with all different classes of anti-cancer drugs tested todate.

GRP78 is a member of the Hsp70 family of heat shock proteins. In normalcells, GRP78 is localized predominantly in the endoplasmic reticulum(EndRet), where it facilitates the correct folding and assembly ofproteins, including the translocation across the ER membrane and thetargeting of misfolded proteins for degradation. See Sitia, R. and I.Braakman, Quality control in the endoplasmic reticulum protein factory.Nature, 2003. 426(6968): p. 891-4 and Xu, C., B. Bailly-Maitre, and J.C. Reed, Endoplasmic reticulum stress: cell life and death decisions. JClin Invest, 2005. 115(10): p. 2656-64. Normal unstressed cells have lowlevels of misfolded proteins and express low basal levels of GRP78.Under conditions of stress, higher levels of misfolded proteins aregenerated and the unfolded protein response (UPR) is activated. The UPRis mediated through three EndRet transmembrane receptors: protein kinaseRNA-like endoplasmic reticulum kinase (PERK), activating transcriptionfactor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1). In unstressedcells, all three ER stress chaperones are maintained in an inactive formby the binding of GRP78. See Ma, Y. and L. M. Hendershot, The role ofthe unfolded protein response in tumour development: friend or foe? NatRev Cancer, 2004. 4(12): p. 966-77.

During stress, the number of misfolded proteins increases and GRP78binds to them, releasing these transmembrane proteins, resulting in theinitiation of a cascade of downstream activities including translationattenuation and up-regulation of ER stress target genes. See Lai, E., T.Teodoro, and A. Volchuk, Endoplasmic reticulum stress: signaling theunfolded protein response. Physiology (Bethesda), 2007. 22: p. 193-201.Through these functions, GRP78 is a master regulator of cell survivalunder conditions of stress.

In vivo models show that homozygous GRP78 (−/−) knock-outs areembryonically lethal, while heterozygous GRP78 (+/−) knock-out micedevelop and function normally. These data suggest that some GRP78 isrequired for embryogenesis but normal cells can tolerate a high degreeof GRP78 down-regulation without adverse effects. See Luo, S., et al.,GRP78/BiP is required for cell proliferation and protecting the innercell mass from apoptosis during early mouse embryonic development. MolCell Biol, 2006. 26(15): p. 5688-97.

In tumor cells, GRP78 assumes the role of a key tumor cell survival andresistance factor. GRP78 in cancer cells differs from normal cells inthat GRP78 levels are significantly higher in tumor cells than in normalstressed cells, and the pattern of GRP78 localization differs from thatof normal stressed cells. Unlike normal cells where GRP78 remains mainlyconfined to the EndRet, tumor cells have significant levels of GRP78 inthe cytoplasm, nucleus, mitochondria, and cell surface. In addition,tumor cells secrete GRP78 into the peritumoral milieu. The combinationof the increased levels and aberrant localization of GRP78 in cancercells gives rise to increased tumor cell proliferation, Elevated GRP78expression levels in tumors has been shown in a wide variety of cancertypes including lung, gastric, breast, hepatocellular, thyroid,melanoma, glioma, colorectal, pancreatic, bladder and various leukemias(Table 1). In these tumor types, the method for detection of GRP78 werevariable, utilizing immunohistochemistry (IHC) analysis, western blotanalysis for GRP78 protein levels, northern blot analysis, or RT-PCR forGRP78 mRNA levels in either tumor derived cell lines or in patient tumorspecimens.

In the tumor biopsy studies, GRP78 expression level in tumor cells waselevated compared to adjacent non-cancerous tissue.

In a hepatocellular carcinoma (HCC) study, GRP78 mRNA was significantlyhigher in 11 of 13 HCC tissues compared to the adjacent non-canceroustissues (p<0.05) [14]. In addition, the sensitivity of HCC cells tosorafenib is correlated to level of GRP78 as determined by GRP78 siRNAexperiments. See Chiou, J. F., et al., Glucose-regulated protein 78 is anovel contributor to acquisition of resistance to sorafenib inhepatocellular carcinoma. Ann Surg Oncol, 2010. 17(2): p. 603-12.

In brain tumors, IHC and Western blot studies reveal that GRP78 issignificantly elevated in malignant glioma specimens and human malignantglioma cell lines, compared to normal adult brain. The studies alsoshowed high GRP78 levels correlated with increased rate of tumor cellproliferation. See Pyrko, P., et al., The unfolded protein responseregulator GRP78/BiP as a novel target for increasing chemosensitivity inmalignant gliomas. Cancer Res, 2007. 67(20): p. 9809-16 and Virrey, J.J., et al., Stress chaperone GRP78/BiP confers chemoresistance totumor-associated endothelial cells. Mol Cancer Res, 2008. 6(8): p.1268-75.

In a melanoma study using fresh biopsy isolates, melanoma tumor cellswere shown to express elevated GRP78 compared with normal melanocytes.Furthermore, the fresh melanoma tumor isolates had up to 4 times greaterlevels of GRP78 by Western blot compared to cultured melanoma celllines. See Jiang, C. C., et al., Glucose-regulated protein 78antagonizes cisplatin and adriamycin in human melanoma cells.Carcinogenesis, 2009. 30(2): p. 197-204.

In a breast cancer study, approximately 65% of pretreatment tumorspecimens expressed high levels of GRP78 by IHC. See Lee, E., et al.,GRP78 as a novel predictor of responsiveness to chemotherapy in breastcancer. Cancer Res, 2006. 66(16): p. 7849-53. This agrees with aprevious published report by Fernandez, et al, which demonstrated a 1.8to 20 fold overexpression of GRP78 mRNA in ⅗ estrogen receptor positivebreast tumors and 6/9 estrogen receptor negative breast tumors comparedto 0/5 benign breast lesions. See Fernandez, P. M., et al.,Overexpression of the glucose-regulated stress gene GRP78 in malignantbut not benign human breast lesions. Breast Cancer Res Treat, 2000.59(1): p. 15-26.

In a study of thyroid cancer, Wang et al showed thyroid cancer cellsexpress high basal levels of GRP78 as assessed by real-time RT-PCR andWestern blot. In addition, the sensitivity of thyroid cancer cells toproteosome inhibition is correlated to the level of GRP78 as determinedby GRP78 siRNA experiments. Wang, H. Q., et al., Different induction ofGRP78 and CHOP as a predictor of sensitivity to proteasome inhibitors inthyroid cancer cells. Endocrinology, 2007. 148(7): p. 3258-70.

Correlation of high GRP78 expression level in tumor biopsy with poorsurvival has been shown in gastric and colorectal cancers. See Xing, X.,et al., Overexpression of glucose-regulated protein 78 in colon cancer.Clin Chim Acta, 2006. 364(1-2): p. 308-15. Zhang, et al., report IHCanalysis of biopsies from 86 patients with primary gastric cancerdemonstrating that GRP78 was overexpressed in the tumor cells whencompared with the adjacent tumor-free gastric mucosa. See Zhang, J., etal., Association of elevated GRP78 expression with increased lymph nodemetastasis and poor prognosis in patients with gastric cancer. Clin ExpMetastasis, 2006. 23(7-8): p. 401-10. The intensity of tumor GRP78staining was graded as negative, weak or strong. The level of GRP78expression levels showed a significant correlation with median overallsurvival with median survival for patients whose tumors stained asnegative, weak or strong of 2489, 1242, and 432 days, respectively(p<0.001 for overall survival of negative versus strong GRP78 tumorexpression). Similarly, GRP78 expression in lymph nodes correlated withpoor overall survival (p=0.037 for overall survival of negative versusany GRP78 expression in lymph nodes).

In a more recent study, Tsunemi, et al, assessed the localization ofGRP78 expression in gastric cancer tissue and normal gastric mucosa byIHC. In normal gastric mucosa, GRP78 staining was occasionally observedin the deep propria glands, but not in the superficial epithelium. Ingastric cancer tissue, GRP78 was expressed at high levels in thecytoplasm of cancer cells regardless of the depth from the surface. Inthe same study, circulating GRP78 protein was assessed in the serum ofboth patients with gastric cancer and normal individuals. Western blotsagainst recombinant GRP78 showed reactivity in sera from 17/60 (28.3%)patients with gastric cancer and 0/20 (0.0%) of healthy individuals. SeeTsunemi, S., et al., Proteomics-based identification of atumor-associated antigen and its corresponding autoantibody in gastriccancer. Oncol Rep, 2010. 23(4): p. 949-56.

IT-139 was selected for its activity in various resistant tumor celllines, and therefore its target(s) were expected to be those that affectresistance. The primary target of IT-139 has now been identified to beGRP78. It has now been found that, surprisingly, IT-139 suppresses thestress up-regulation of GRP78 in tumor cells. This effect is specific totumor cells, as IT-139 does not affect GRP78 expression in normal cells.Treatment of normal cells under non-stressed and stressed conditionswith IT-139, showed that: 1) IT-139 does not effect the basal GRP78levels in non-stressed normal cells; and 2) IT-139 does not effect GRP78up-regulation due to stress in these same normal cells. Therefore it isbelieved that IT-139 does not impact GRP78 levels in normal cellsregardless of stress conditions.

Without wishing to be bound by any particular theory, it is believedthat IT-139 is not a general inhibitor of the UPR but rather a specificsuppressor of GRP78 induction. The main pathway of GRP78 induction isvia transcription. IT-139 suppression of GRP78 is at the transcriptionallevel in a dose dependent manner, as seen by Northern blot analysis oftumor cells treated with IT-139. In some embodiments, the presentinvention encompasses the finding that IT-139 suppresses the inductionof GRP78 by stress inducing agents.

It was surprisingly found that IT-139 does not block other arms of theUPR such as induction XBP-1 spliced form, induction, processing andnuclear import of ATF6, and phosphorylation of eIF2a. IT-139 thereforecauses ER stress and part of the UPR, but suppresses induction of GRP78(the survival arm of UPR).

The IT-139 suppression of GRP78 induction at the transcriptional levelis confirmed by GRP78 promoter studies. Regulation of GRP78 proteinlevels in the cell is primarily via transcriptional control due to thefact that the GRP78 promoter contains multiple copies of endoplasmicreticulum stress elements (ERSE). ERSEs are binding sites of the stressinduced transcription factors. IT-139 has been to shown to suppressstress-induction of the GRP78 promoter fragment (−169 to −29; contains 3ERSEs) linked to a luciferase reporter gene.

Induction of GRP78 is the cell's survival response under conditions ofstress. It is the attempt of the cell to repair itself and preventapoptosis. GRP78 induction is therefore seen when cells arestressed/dying. IT-139 suppression of GRP78 in tumor cells is mostprominent in stressed tumor cells. Tumor cells in vivo are alwaysundergoing various kinds of stress. In non-stressed tumor cells invitro, IT-139 suppresses GRP78 levels to varying levels in differenttumor lines.

High levels of GRP78 protein have been correlated with resistance toagents such as cisplatin (Jiang, C. C., et al., Glucose-regulatedprotein 78 antagonizes cisplatin and adriamycin in human melanoma cells.Carcinogenesis, 2009. 30(2): p. 197-204), 5-FU (Pyrko, P., et al., Theunfolded protein response regulator GRP78/BiP as a novel target forincreasing chemosensitivity in malignant gliomas. Cancer Res, 2007.67(20): p. 9809-16), temozolomide (Pyrko, 2007), vinblastine (Wang, J.,et al., Blockade of GRP78 sensitizes breast cancer cells tomicrotubules-interfering agents that induce the unfolded proteinresponse. J Cell Mol Med, 2009. 13(9B): p. 3888-97), paclitaxel(Mhaidat, N. M., et al., Inhibition of MEK sensitizes paclitaxel-inducedapoptosis of human colorectal cancer cells by downregulation of GRP78.Anticancer Drugs, 2009. 20(7): p. 601-6), bortezomib (Kern, J., et al.,GRP-78 secreted by tumor cells blocks the antiangiogenic activity ofbortezomib. Blood, 2009. 114(18): p. 3960-7), sorafenib (Chiou, J. F.,et al., Glucose-regulated protein 78 is a novel contributor toacquisition of resistance to sorafenib in hepatocellular carcinoma. AnnSurg Oncol, 2010. 17(2): p. 603-12), camptothecin (Reddy, R. K., et al.,Endoplasmic reticulum chaperone protein GRP78 protects cells fromapoptosis induced by topoisomerase inhibitors: role of ATP binding sitein suppression of caspase-7 activation. J Biol Chem, 2003. 278(23): p.20915-24), etoposide (Wang, Y., et al., Down-regulation of GRP78 isassociated with the sensitivity of chemotherapy to VP-16 in small celllung cancer NCI-H446 cells. BMC Cancer, 2008. 8: p. 372) and doxorubicin(Jiang, C. C., et al., Glucose-regulated protein 78 antagonizescisplatin and adriamycin in human melanoma cells. Carcinogenesis, 2009.30(2): p. 197-204). Furthermore, treatment of tumor cell lines withseveral of these agents further up-regulates levels of GRP78 protein.See Jiang 2009 and Reddy 2003. This additional up-regulation of GRP78induced by anticancer agents is thought to be a significant determinantof tumor cell survival and resistance. That IT-139 preferentiallyprevents GRP78 induction in “stressed” tumor cells, suggested thatIT-139 would be synergistic when used in combination with anti-canceragents of many different classes.

Multiple GRP78 transcription factors are effected following stressinduction, including NF-Y, TFII-I, ATF6α, and YY-1. NF-Y binding ispreserved in stressed and non-stressed GRP78 transcription. TFII-Ibinding is enhanced in stressed transcription. ATF6 is cleaved to ATF6αwithin 1 h of thapsigargin (Tg) stress treatment and results only afterER stress. This complex (ATF6α/YY1) recruits PRMT1 to the promoter alongwith methylated histone H4, p300, GCN5 and histone acetyltransferases.ATF6α functions (at least in part) by recruiting a collection of RNApolymerase II coregulatory complexes, including the Mediator andmultiple histone acetyltransferase complexes (Spt-Ada-Gcn5acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes) tothe ER stress response enhancer elements. Without wishing to be bound toany particular theory, we propose that IT-139 inhibits to loading ofthis POL II complex on the GRP78 promoter region.

One embodiment of the present invention is that IT-139's mechanism ofaction is an effect on the transcription of GRP78. Another embodiment ofthe present invention is that IT-139 inhibits the stress-inducedtranscription of GRP78. Transcriptional activation of GRP78 is anindicator of the unfolded protein response. UPR induces specificacetylation and methylation modification of nucleosomes. It is theorizedthat the ERSE is the most critical element mediating the stressinduction of the GRP78 promoter.

Another aspect of the present invention is a method of treating a cancerin a subject in need thereof, comprising administering IT-139, or apharmaceutically acceptable composition thereof, in combination with oneor more immuno-oncology therapeutics. Tumor-borne ER stress imprints abinitio BMDC to a phenotype that recapitulates several of theinflammatory/suppressive characteristics ascribed to tumor-infiltratingmyeloid cells, highlighting the tumor UPR as a critical controller ofanti-tumor immunity and a new target for immune modulation in cancer.(See Mahadevan et al. PlosONe December 2012) Shedding of the NKG2Dligand, MICA, by chronic lymphocytic leukemia cells can be induced upontranslocation of the endoplasmic reticulum-resident proteins ERp5 andGRP78 to the tumor cell surface. (See Cancer Immunol Immunother (2012)61:1201) Surface LAP/TGF-β forms a complex with GRP78, and knockdown ofGRP78 reduces the expression levels of surface LAP/TGF-β on Tregs. (SeeHum. Immunol. 62, 764-770, 2001) Therefore, without wishing to be boundto any theory, we believe that combination therapy comprising IT-139 andan immuno-oncology agent will result in a more effective treatment thanthe immuno-oncology agent alone.

2. Definitions

As described herein, the phrase immuno-oncology agent refers to anycancer immunotherapy agent wherein the immune system is leveraged totreat cancer. Such agents include, but are not limited to, antibodies,PD-1 therapies, PD-L1 therapies, cytokine therapeutics, and checkpointinhibitors. Specific examples include, but are not limited to,nivolumab, alemtuzumab, atezolizumab, ipilimumab, ofatumumab,pembrolizumab, rituximab, interferon, and interleukin. Targets ofimmune-oncology agents include, but are not limited to, CD52, PD-L1,CTLA4, CD20, or the PD-1 receptor.

As described herein, the phrase chemotherapy agent or chemotherapeuticagent describes a chemical substance used to treat cancer. Such agentsinclude cytotoxic and cytostostatic drugs. A chemotherapy agent orchemotherapeutic agent may also refer to an antibody or a monoclonalantibody (MAB). Classes of chemotherapeutic agents include, but are notlimited to: taxanes, anthracyclines, platinum containing drugs,epothilones, anti-mitotic agents, camptothecins, folic acid derivatives,HDAC inhibitors, mitotic inhibitors, microtubule stabilizers, DNAintercalators, topoisomerase inhibitors, or molecularly targetedtherapeutics. The phrase chemotherapy agent or chemotherapeutic agentmay also refer to one or more chemical substances combined together totreat cancer. One non-limiting example of this may include gemcitabineand nanoparticle albumin paclitaxel.

As used herein, the term IT-139 refers to sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate(III)]. IT-139 is also knownas KP1339 or NKP1339.

Vinca alkaloids are well known in the literature and are a set ofanti-mitotic agents. Vinca alkaloids include vinblastine, vincristine,vindesine, and vinorelbine, and act to prevent the formation ofmicrotubules. Exemplary vinca alkaloids are shown below.

The antitumor plant alkaloid camptothecin (CPT) is a broad-spectrumanticancer agent that targets DNA topoisomerase I. Although CPT hasshown promising antitumor activity in vitro and in vivo, it has not beenclinically used because of its low therapeutic efficacy and severetoxicity. Among CPT analogues, irinotecan hydrochloride (CPT-11) hasrecently been shown to be active against colorectal, lung, and ovariancancer. CPT-11 itself is a prodrug and is converted to7-ethyl-10-hydroxy-CPT (known as SN-38), a biologically activemetabolite of CPT-11, by carboxylesterases in vivo. A number ofcamptothecin derivatives are in development, the structures of which areshown below.

Several anthracycline derivates have been produced and have found use inthe clinic for the treatment of leukemias, Hodgkin's lymphoma, as wellas cancers of the bladder, breast, stomach, lung, ovaries, thyroid, andsoft tissue sarcoma. Such anthracycline derivatives include daunorubicin(also known as Daunomycin or daunomycin cerubidine), doxorubicin (alsoknown as DOX, hydroxydaunorubicin, or adriamycin), epirubicin (alsoknown as Ellence or Pharmorubicin), idarubicin (also known as4-demethoxydaunorubicin, Zavedos, or Idamycin), and valrubicin (alsoknown as N-trifluoroacetyladriamycin-14-valerate or Valstar).

Platinum based therapeutics are well known in the literature. Platinumtherapeutics are widely used in oncology and act to crosslink DNA whichresults in cell death (apoptosis). Carboplatin, picoplatin, cisplatin,and oxaliplatin are exemplary platinum therapeutics and the structuresare shown below.

Additional molecularly targeted therapeutics are also in development.Examples include E7016, XL765, TG101348, E7820, eribulin, INK 128,TAK-385, MLN2480, TAK733, MLN-4924, motesanib, ixazomib, TAK-700,dacomitinib, and sunitinib. The structures of each are shown below.

Further examples of molecularly targeted therapeutics includecrizotinib, axitinib, PF 03084014, PD 0325901, PF 05212384, PF 04449913,ridaforlimus, MK-1775, MK-2206, GSK2636771, GSK525762, eltrombopag,dabrefenib, and foretinib. The structures of each are shown below.

Yet further examples of molecularly targeted therapeutics includelapatinib, pazopanib, CH5132799, R04987655, RG7338, A0379, erlotinib,pictilisib, GDC-0032, venurafenib, GDC-0980, GDC-0068, arry-520,pasireotide, dovitinib, and cobmetinib. The structures of each are shownbelow.

Additional examples of molecularly targeted therapeutics includebuparlisib, AVL-292, romidepsin, arry-797, lenalidomide, thalidomide,apremilast, AMG-900, AMG208, rucaparib, NVP-BEZ 235, AUY922, LDE225, andmidostaurin. The structures of each are shown below.

3. Description of Exemplary Embodiments

The present invention provides a method for treating cancer in a patientin need thereof comprising administering IT-139, or a pharmaceuticallyacceptable composition thereof, in combination with a chemotherapeuticagent or an immuno-oncology agent.

According to another embodiment, the present invention relates to amethod of treating a cancer selected from breast, ovary, cervix,prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, myeloid disorders, lymphoiddisorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, and leukemia,comprising administering IT-139, or a pharmaceutically acceptablecomposition thereof, in combination with a chemotherapeutic agent or animmuno-oncology agent.

Another embodiment provides a method for treating cancer by reducing theamount of GRP78 in cancer cells following administration of IT-139.

According to another embodiment, the present invention provides a methodfor treating cancer by reducing the amount of GRP78 in cancer cellsfollowing administration of IT-139 in combination with a chemotherapyagent or an immune-oncology agent, wherein the administration of IT-139,or a pharmaceutically acceptable composition thereof, results in areduction in the amount of GRP78 as compared to administration of thechemotherapy agent or immune-oncology agent alone.

The order of administration of therapeutics should be carefullyconsidered. Without wishing to be bound to any particular theory, themechanism of action and down-regulation of GRP78 dictates that anychemotherapeutic agent should be administered first, followed by IT-139for maximum therapeutic benefit. As stated above, treatment with a rangeof chemotherapeutic agents results in an increase ER stress, whichinduces production of GRP78. This process is a cellular survivalmechanism. Administration of IT-139 decreases the level ofstress-induced GRP78, which removes a cellular survival pathway. Theultimate result is increased cancer cell death and increased anti-tumoreffect.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprising the stepsof:

-   -   1) administering to the patient a chemotherapy agent;    -   2) subsequently administering IT-139, or a pharmaceutically        acceptable composition thereof; to the patient; and    -   3) optionally repeating steps 1 and 2.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered 1 day after the chemotherapy agent.In other embodiments, IT-139, or a pharmaceutically acceptablecomposition thereof, is administered to the patient 1 week after thechemotherapy agent. In yet other embodiments, IT-139 is administered toa patient between 1 and seven days after the chemotherapy agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered simultaneously with thechemotherapy agent. In certain embodiments, the IT-139, or apharmaceutically acceptable composition thereof, and the chemotherapyagent are administered within about 20-28 hours of each other, or withinabout 22-26 hours of each other, or within about 24 hours of each other.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before the chemotherapy agent. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours beforethe chemotherapy agent, or at least about 10-14 hours before thechemotherapy agent, or at least about 12 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours beforethe chemotherapy agent, or at least about 22-26 hours before thechemotherapy agent, or at least about 24 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours beforethe chemotherapy agent, or at least about 46-50 hours before thechemotherapy agent, or at least about 48 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours beforethe chemotherapy agent, or at least about 70-74 hours before thechemotherapy agent, or at least about 72 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before the chemotherapy agent. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours after thechemotherapy agent, or at least about 10-14 hours after the chemotherapyagent, or at least about 12 hours after the chemotherapy agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours afterthe chemotherapy agent, or at least about 22-26 hours after thechemotherapy agent, or at least about 24 hours after the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours afterthe chemotherapy agent, or at least about 46-50 hours after thechemotherapy agent, or at least about 48 hours after the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours afterthe chemotherapy agent, or at least about 70-74 hours after thechemotherapy agent, or at least about 72 hours after the chemotherapyagent.

In certain embodiments, the chemotherapeutic agent is selected from thegroup consisting of gemcitabine, nanoparticle albumin paclitaxel,paclitaxel, docetaxel, cabazitaxel, oxaliplatin, cisplatin, carboplatin,doxorubicin, daunorubicin, sorafenib, everolimus and vemurafenib. Incertain embodiments, the chemotherapeutic agent is gemcitabine.

According to one embodiment of the present invention provides a methodfor treating pancreatic cancer in a patient in need thereof, comprisingthe steps of:

-   -   1) administering a gemcitabine and albumin nanoparticle        paclitaxel;    -   2) subsequently administering IT-139, or a pharmaceutically        acceptable composition thereof; and    -   3) optionally repeating steps 1 and 2.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered simultaneously with gemcitabine. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, and gemcitabine are administered within about 20-28hours of each other, or within about 22-26 hours of each other, orwithin about 24 hours of each other.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before gemcitabine. In certainembodiments, the IT-139, or a pharmaceutically acceptable compositionthereof, is administered at least about 8-16 hours before gemcitabine,or at least about 10-14 hours before gemcitabine, or at least about 12hours before gemcitabine.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours beforegemcitabine, or at least about 22-26 hours before gemcitabine, or atleast about 24 hours before gemcitabine.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours beforegemcitabine, or at least about 46-50 hours before gemcitabine, or atleast about 48 hours before gemcitabine.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with an immuno-oncology agent. In certainembodiments, the immune-oncology agent is administered to the patientprior to the administration of IT-139, or a pharmaceutically acceptablecomposition thereof.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered simultaneously with theimmuno-oncology agent. In certain embodiments, the IT-139, or apharmaceutically acceptable composition thereof, and the immuno-oncologyagent are administered within about 20-28 hours of each other, or withinabout 22-26 hours of each other, or within about 24 hours of each other.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before the immuno-oncology agent.In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours beforethe immuno-oncology agent, or at least about 10-14 hours before theimmuno-oncology agent, or at least about 12 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours beforethe immuno-oncology agent, or at least about 22-26 hours before theimmuno-oncology agent, or at least about 24 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours beforethe immuno-oncology agent, or at least about 46-50 hours before theimmuno-oncology agent, or at least about 48 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours beforethe immuno-oncology agent, or at least about 70-74 hours before theimmuno-oncology agent, or at least about 72 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered after the immuno-oncology agent. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours after theimmuno-oncology agent, or at least about 10-14 hours after theimmuno-oncology agent, or at least about 12 hours after theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours afterthe immuno-oncology agent, or at least about 22-26 hours after theimmuno-oncology agent, or at least about 24 hours after theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours afterthe immuno-oncology agent, or at least about 46-50 hours after theimmuno-oncology agent, or at least about 48 hours after theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours afterthe immuno-oncology agent, or at least about 70-74 hours after theimmuno-oncology agent, or at least about 72 hours after theimmuno-oncology agent.

In certain embodiments, the immune-oncology agent is selected from thegroup consisting of cytokines, checkpoint inhibitors and antibodiesother than PD-1 antibodies. In certain embodiments, the immune-oncologyagent is selected from the group consisting of interferon, interleukin,PD-L1 antibodies, alemtuzumab, ipilimumab, ofatumumab, atezolizumab andrituximab.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with a PD-1 antibody. In certain embodiments,the PD-1 antibody is administered prior to the administration of theIT-139, or a pharmaceutically acceptable formulation thereof.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with a PD-L1 antibody. In certain embodiments,the PD-L1 antibody is administered prior to the administration of theIT-139, or a pharmaceutically acceptable formulation thereof.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with an immune-oncology agent other than a PD-1antibody. In certain embodiments, the immune-oncology agent other than aPD-1 antibody is administered prior to the administration of the IT-139,or a pharmaceutically acceptable formulation thereof.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It will be understoodthat these examples are for illustrative purposes only and are not to beconstrued as limiting this invention in any manner.

EXEMPLIFICATION Example 1

All human primary and metastatic cell lines were maintained as describedby ATCC. Cells were seeded in a 6 well plate prior to treatment withmedia alone (control), IT-139 (200 μM) alone, thapsigargin alone (300nM, to induce cell stress) or IT-139 plus thapsigargin. Cell lysateswere collected at 16 hours and were analyzed by western blot foranti-GRP78 antibody and normalized by GAPDH. The results shown in FIG. 1demonstrate that IT-139 had little to no effect on unstressed cells(FIG. 1A) while treatment with IT-139 in stressed cells (IT-139 plusthapsigargin) decreased the amount of GRP78 present in stressed cells(FIG. 1B).

Example 2

All human primary and metastatic cell lines were maintained as describedby ATCC. Cells were seeded in a 6 well plate prior to treatment withmedia alone (control), IT-139 (200 μM) alone, thapsigargin alone (300nM, to induce cell stress) or IT-139 plus thapsigargin. Cell lysateswere collected at 24 hours and total RNA was extracted. Quantitativereal-time PCR analysis of GRP78 transcripts normalized to GAPDH. Theresults shown in FIG. 2 demonstrate that IT-139 treatment in unstressedcells generally has little impact on grp78 mRNA levels (FIG. 2A) whiletreatment with IT-139 in stressed cells (IT-139 plus thapsigargin)decreased the amount of GRP78 mRNA expression (FIG. 2B).

Example 3

CT26 cells (1 million) were subcutaneously implanted in immunocompetentmice and allowed to grow until a palpable tumor was established (day 3).Groups of four mice per group were treated twice weekly with four totaldoses of either 1) saline, 2) IT-139 alone (KP1339, 30 mg/kg), 3)RPM1-14 (PD-1 antibody, 5 mg/kg), or 4) RPM1-14 and IT-139 (5 mg/kg and30 mg/kg, respectively). IT-139 was administered intravenously andRPM1-14 was administered intraperitoneally. Administrations were made onthe same day. Tumor volume was measured through day 18. Treatment withthe PD-1 antibody showed no change from saline control. IT-139demonstrated anti-tumor activity, however the combination of PD-1antibody and IT-139 demonstrated increased antitumor activity comparedto either PD-1 antibody alone or IT-139 alone. The results shown in FIG.3 demonstrate that IT-139 increases the anti-tumor efficacy of a PD-1antibody.

Example 4

HCT116 cell line was seeded one day prior to treatment according to ATCCguidelines. 24 hours after treatment with IT-139 (200 μM), treated anduntreated cells were fixed for electron microscopic evaluation. As shownin FIG. 4, HCT116 cells treated with IT-139 (FIG. 4B) showed significantvacuolization, ER expansion and disorganization of intracellularorganelles suggesting ER stress when compared to untreated cells (FIG.4A).

Example 5

Chromatin immunoprecipitation (ChIP) assay was performed to examinePolymerase II binding to Pol II and GRP78 promoter regions in stressedand unstressed cells with IT-139 treatment. HCT116 cells were grown to80% confluence then treated with 1.5 μg/mL tunicamycin and 200 μM IT-139or DMSO for 16 hours. Chromatin was cross-linked using formaldehyde.Cells were harvested with trypsin and isolated nuclei were sonicated toyield fragments between 200-1000 bp. Equal amounts of chromatin wereincubated with anti-Pol II antibody overnight then pulled down withStaph A cells. Cross-linking was reversed and the DNA was purified usinga GeneElute PCR cleanup kit from Sigma. Purified DNA and input sampleswere subjected to 30 cycles of PCR with primers amplifying promoterregions of Grp78 and Pol II. The products were run on a 4% agarose geland visualized with ethidium bromide staining. IT-139 had a minimaleffect on Polymerase II binding to the Pol II promoter in non-stressedcells, but reduced this binding to 40% in Tu-stressed cells. Strikingly,IT-139 dropped Polymerase II binding to the Grp78 promoter to zero inboth non-stressed and Tu-stressed cells. These results are shown in FIG.5A-C.

Example 6

Immunohistochemical analysis was performed to analyze the expression ofGRP78 in HT-29 xenograft tumors treated with IT-139 at 30 mg/kg (q4d) incomparison to saline treated tumors. Strong immunostaining of GRP78 wasobserved in saline treated tumors (FIG. 6A) in comparison to very weakstaining in IT-139 treated tumors (FIG. 6B). These results indicatedthat IT-139 inhibits GRP78 expression in vivo.

Example 7

Normal human embryonic kidney 293T cells were either non-stressed(treated with normal media) or stressed (treated with 300 nM EndRetstress & GRP78 inducer Thapsigargin). Cell cultures were either treatedwith no drug (control) or 200 μM IT-139. GRP78 levels assayed by westernblot; β-actin is loading control. The results are shown in FIG. 7.

Example 8

Prostate cancer LnCaP-FGC cells were untreated (DMSO only control) ortreated with 300 nM thapsigargin (Tg). Cells were co-incubated with theindicated concentrations of IT-139 and Northern blot analysis wasperformed on cell lysates. β-actin is the loading control. Figure showsthe Northern blots. Bands were quantified and bar graph shows relativelevels of GRP78 mRNA levels normalized against β-actin loading control.IT-139 suppression of GRP78 is at the transcriptional level in a dosedependent manner, as seen by Northern blot analysis of tumor cellstreated with IT-139. The results are shown in FIG. 8.

Example 9

All human primary and metastatic cell lines were maintained as describedby ATCC. All cell lines (HCT116, HT-29, LNCaP, A549 and A375) wereseeded in a 6 well plate 24 hours prior to treatment with IT-139 alone.All cell lines were treated with 30 uM, 50 uM, 100 uM and 200 uM withthe following exceptions: treatment with 30 uM and 200 uM were omittedfor HCT116 and HT29 cell lines respectively. After 24 h incubation withthe drug, cells were trypsinized and re-suspended in warm PBS andstained with the JC-1 dye for 30 min at 37 C in the dark. The cells werethen washed and re-suspended in warm PBS. The fluorescence of the JC-1dye was measured by flow cytometry analysis by exciting the dye at 488nm and detecting the JC-1 monomer through its emission at 530 nm withaggregates of JC-1 being measured at 580 nm. All cell lines treated withAntimycin A at 50 uM as positive control. Results show that both HCT116and HT29 cell lines showed increased loss of mitochondrial potential atlower concentrations, 50 μM and 30 μM respectively, of IT-139. However,prostate (LNCaP), lung (A549) and melanoma (A375) cells showed anincrease in mitochondrial depolarization at high concentrations ofIT-139. Data is shown in FIG. 9.

Example 10

Prostate cancer cell line, MiaPaca2, was maintained as described byATCC. MiaPaca2 cells co-cultured with normal peripheral bloodmononuclear cells (PBMCs) treated with increasing concentrations ofIT-139 (50, 100 and 200 μM) for 24 h did not show any effect upon cellviability as shown in FIG. 10. MiaPaca2 cells were either untreated orpre-treated with IT-139 (100 μM) and co-cultured with IL-2 activatedPBMCs at varying doses. PBMCs were activated using 6000 IU of IL-2 for24 h. IT-139 shows increased cell death in MiaPaca2 cells co-culturedwith activated PBMCs, as shown in FIG. 11.

Example 11

Combination IT-139 and gemcitabine (GEM) treatment extend both medianand overall survival in an ASPC mouse model. Likewise, combinationdosing at 48 hours of Gemcitabine in combination with IT-139 in ASPC-1cells in vitro, results in a significant change in survival when IT-139is dosed first. In vitro ASPC20 cells are treated for 48 hours with DMSO(control), 150 μM IT-139, 5 μM gemcitabine, simultaneous 150 μM IT-139and 5 μM gemcitabine, 5 μM gemcitabine for 24 hours followed by 150 μMIT-139, or 150 μM IT-139 followed by 24 hrs 5 μM gemcitabine. Cells areharvested and counted by trypan blue to calculate the number of deadcells versus viable cells. In ASPC20 cells, the order of dosingthapsigargin (Tg) to induce stress in combination with IT-139, affectslevels of GRP78 expression at 30 hours. The results are shown in FIG.12. In addition, the order of dosing also effects the amount of celldeath, as increased cell death is observed when IT-139 is dosed beforegemcitabine. The results are shown in FIG. 13. PANC-1 cells are treatedin vitro for 48 hours with DMSO (control), 150 μM IT-139, 5 μMgemcitabine, simultaneous 150 μM IT-139 and 5 μM gemcitabine, 5 μMgemcitabine for 24 hours followed by 150 μM IT-139, and 150 μM IT-139followed by 24 hours 5 μM gemcitabine DMSO (control), 150 μM IT-139, 5μM gemcitabine, simultaneous 150 μM IT-139 and 5 μM gemcitabine, 5 μMgemcitabine for 24 hours followed by 150 μM IT-139, and 150 μM IT-139followed by 24 hours 5 μM gemcitabine. The results are shown in FIG. 14.In PANC-1 cells, the cytoxicity effect at 48 hours demonstrates nodifference dependent on sequence of dosing of gemcitabine and IT-139.

Example 12

In an A20 lymphoma mouse model, 104 Balb/c mice were inoculated withlymphoma A20 cells subcutaneously on the right flank of the mice. Micewere randomized into 8 groups of 10 mice when tumors reached a meanvolume of 80-120 mm³. Mice were monitored daily for behavior andsurvival and twice weekly for body weight and tumor growth. Tumors wereinduced by subcutaneous injection of 5×10⁶ of A20 cells in 200 μL RPMI1640 medium containing matrigel (50:50, v:v, ref: 356237, BDBiosciences, France) into the right flank of hundred and four (104)Balb/C mice. The checkpoint inhibitors were Anti-PD-L1 (clone 10F.9G2;ref: Bioxcell isoptype Rat IgG2b), and Anti-CTLA4 antibody (clone 9H10;ref: BE0131, bioxcell; isotype Hamster IgG1), both dosed at 10 mg/kgi.p. every 3 days. IT-139 was dosed at 30 mg/kg intravenously every 4days. The dosage schedule is shown in Table 1 below. Average tumorvolume over the course of the experiment is shown in FIG. 15. 5 tumorsfrom each group were collected for FACS Analysis (40 tumor samples). Onepanel was run for Treg and T effector cells (CD45, CD3, CD4, CD8 andFoxP3), one panel for MDSCs (CD45, CD3, CD11b, Gr-1, Ly-6g, Ly-6C, Arg1,NOS2), and one panel for tumor-associated macrophages (CD54, CD11b,Gr-1, CD68, CD80, and CD206). When PD-L1 was dosed 24 hours prior toIT-139 administration there was a significant anti-tumor efficacy and a10% increase in effector T-Cell infiltration of the tumor, shown in FIG.16 and FIG. 17. This effect was not seen in any of the other groups.With the decrease in tumor growth in this combination group, evidence ofan immunomodulatory effect with anti-PD-L1 antibody is present.

TABLE 1 A20 Immunotherapy Study Design and Grouping Dose (mg/ No. kg/Group Animals Treatment inj) Route Treatment schedule 1 10 Vehicle — IVD_(R), D_(R+3), D_(R+7), D_(R+10) 2 10 IT-139 30 IV D_(R), D_(R+3),D_(R+7), D_(R+10) 3 10 Anti- 10 IP D_(R+1), D_(R+4), D_(R+8), D_(R+11)CTLA-4 4 10 Anti-PD-L1 10 IP D_(R+1), D_(R+4), D_(R+8), D_(R+11) 5 10IT-139 30 IV D_(R), D_(R+3), D_(R+7), D_(R+10) Anti- 10 IP D_(R+1),D_(R+4), D_(R+8), D_(R+11) CTLA-4 6 10 IT-139 30 IV D_(R+2), D_(R+5),D_(R+9), D_(R+12) Anti- 10 IP D_(R+1), D_(R+4), D_(R+8), D_(R+11) CTLA-47 10 IT-139 30 IV D_(R), D_(R+3), D_(R+7), D_(R+10) Anti-PD-L1 10 IPD_(R+1), D_(R+4), D_(R+8), D_(R+11) 8 10 IT-139 30 IV D_(R+2), D_(R+5),D_(R+9), D_(R+12) Anti-PD-L1 10 IP D_(R+1), D_(R+4), D_(R+8), D_(R+11)TOTAL 80

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example

We claim:
 1. A method for treating pancreatic cancer in a patient inneed thereof comprising administering IT-139, or a pharmaceuticallyacceptable composition thereof, in combination with gemcitabine, andwherein the administration of IT-139, or a pharmaceutically acceptablecomposition thereof, results in a reduction in an amount of GRP78 ascompared to administration of gemcitabine alone.
 2. The method accordingto claim 1, wherein the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered in combination with gemcitabine. 3.The method according to claim 1 or 2, wherein the IT-139, or apharmaceutically acceptable composition thereof, is administered to thepatient after gemcitabine is administered to the patient.
 4. The methodaccording to claim 1, wherein the IT-139, or a pharmaceuticallyacceptable composition thereof, is administered to the patientsimultaneously with the gemcitabine.
 5. The method according to claim 1,wherein the IT-139, or a pharmaceutically acceptable compositionthereof, and the gemcitabine are administered to the patient withinabout 24 hours of each other.
 6. The method according to claim 1,wherein the IT-139, or a pharmaceutically acceptable compositionthereof, is administered to the patient before the gemcitabine isadministered to the patient.
 7. The method according to claim 1, whereinthe IT-139, or a pharmaceutically acceptable composition thereof, isadministered to the patient at least about 12 hours before thegemcitabine is administered to the patient.
 8. The method according toclaim 1, wherein the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered to the patient at least about 24hours before the gemcitabine is administered to the patient.
 9. Themethod according to claim 1, wherein the IT-139, or a pharmaceuticallyacceptable composition thereof, is administered to the patient at leastabout 48 hours before the gemcitabine is administered to the patient.